Human immunoglobulins, essentially containing IgGs, are commonly used in the treatment of pathologies such as immunodeficiency or auto-immune diseases and particularly by intravenous route (IvIG).
Nevertheless, according to clinical observation, the use of IvIGs in patients sometimes results in serious side effects, such as for example thromboembolic events.
An embolic thrombosis consists of the formation of a thrombus obstructing a blood vessel. The thrombus can develop in the venous circulation and give rise to a venous thrombosis, or in the arterial circulation and result in an arterial occlusion with ischaemia or even infarction. A thrombus results from blood coagulation, due to platelet aggregation and the activation of the coagulation system, this being a chain reaction which involves the platelets and the coagulation factors. A thrombus essentially contains fibrin, an insoluble protein formed from fibrinogen.
Factors XI and IX are among the factors involved in intrinsic pathway blood coagulation. Factor IX is activated by activated Factor XI, the latter itself being activated by activated Factor XII. This activation cascade finally results in the formation of fibrinogen. (FI).
The Applicant's studies presented in the present application have made it possible to show that the presence of coagulation factors such as Factor VII, Factor IX, Factor XI, Factor XII or Factor X and/or their activated forms in a human immunoglobulin product such as IvIGs, could be the cause of thromboembolic events after the injection of IvIGs in patients.
As a result, there is a great need to make available a reliable and sensitive process making it possible to determine the presence of activated factor VII, activated Factor XI, activated factor IX, activated Factor XII and/or activated Factor X in a biologically acceptable product containing immunoglobulins.
The thrombin generation test (TGT) is known to a person skilled in the art. The principle of this test relates to analysis of the kinetics of the formation of thrombin that a given plasma produces in response to a standardized stimulation.
Among the TGTs developed to date, thromboelastography consists of measuring the physical properties of whole blood by mechanically analyzing clot formation as a function of time. According to the parameters extracted from a graph (called a thromboelastogram) produced by a thromboelastographer, it is possible to assess a patient's coagulation ability.
Moreover, in 2002, Hemker et al. proposed the concept of thrombinography making it possible to measure the generation of thrombin by fluorometry (Pathophysiol Haemost thromb 2002; 32: 249-53). Thrombinography consists of using a thrombin calibrator and a specific fluorescent substrate in platelet-ploor plasma or in platelet-rich plasma, in order to establish a thrombinogram. A signal induced by the thrombin generated by the tissue factor is confronted with the signal generated by a standardized quantity of exogenous thrombin in this same plasma.
The Applicant of the present application has surprisingly found that a thrombin generation test also makes it possible to determine the presence of FVII, FXI, FIX, FXII, FX and/or of their activated forms in a sample.